New applications of the Comet assay: Comet–FISH and transcription-coupled DNA repair

Abstract

Transcription-coupled repair (TCR) is a pathway dedicated to the removal of damage from the template strands of actively transcribed genes. Although the detailed mechanism of TCR is not yet understood, it is believed to be triggered when a translocating RNA polymerase is arrested at a lesion or unusual structure in the DNA. Conventional assays for TCR require high doses of DNA damage for the statistical analysis of repair in the individual strands of DNA sequences ranging in size from a few hundred bases to 30 kb. The single cell gel electrophoresis (Comet) assay allows detection of single-or double-strand breaks at a 10 to 100-fold higher level of resolution. Fluorescence in situ hybridization (FISH) combined with the Comet assay (Comet-FISH) affords a heightened level of sensitivity for the assessment of repair in defined DNA sequences of cells treated with physiologically relevant doses of genotoxins. This approach also reveals localized susceptibility to chromosomal breakage in cells from individuals with hypersensitivity to radiation or chemotherapy. Several groups have reported preferential repair in transcriptionally active genes or chromosomal domains using Comet-FISH. The prevailing interpretation of the behavior of DNA in the Comet assay assumes that the DNA is arranged in loops and matrix-attachment sites; that supercoiled, undamaged loops are contained within the nuclear matrix and appear in Comet "heads", and that Comet "tails" consist of relaxed DNA loops containing one or more breaks. According to this model, localization of FISH probes in Comet heads signifies that loops containing the targeted sequences are free of damage. This implies that preferential repair as detected by Comet-FISH might encompass large chromosomal domains containing both transcribed and non-transcribed sequences. We review the existing evidence and discuss the implications in relation to current models for the molecular mechanism of TCR.

NSAID-manufacturing plant effluent induces geno- and cytotoxicity in common carp ( Cyprinus carpio )

“…Cytotoxicity is defined as the in situ evaluation of cellular function in response to a toxic substance (Goguen and Kedersha, 1993). DNA lesions in specific sites in the genome may induce changes in the nucleotide sequence via processes such as translesion synthesis or recombination, eliciting mutagenesis and cell responses that may lead to apoptosis (Spivak et al, 2009). Apoptosis is mediated by the group of cysteine proteases known as caspases (Reed et al, 2004).…”

Comet-FISH with strand-specific probes reveals transcription-coupled repair of 8-oxoGuanine in human cells

“…We have previously used the comet-FISH approach with FISH signals localized in the comet head or tail to represent intact or damaged DNA segments, respectively (19). However, lesions induced in a region adjoining the undamaged DNA segment of interest could also result in FISH signals appearing in the comet tail.…”

Toxicogenetic and antioxidant activities of isopentyl ferulate

“…Amount of DNA on heads and tails of comets provides an estimate of frequency of filament breaks. To some lesions types, modified DNA can be directly reverted to original sequence (Spivak et al, 2009), these DNA repair mechanisms allow estimating a possible adaptation to low concentrations of genotoxic element (Widziewicz et al, 2009). In our test, IF showed genotoxicity in peripheral blood and bone marrow cells of mice that was evidenced by the increase in frequency and index of damage at two tested doses with different administration routes (Figures 1 and 2).…”